PROJECT SUMMARY/ABSTRACT My laboratory is dedicated to the development, synthesis, and biochemical validation of small molecules and peptides that quantitate and regulate human and bacterial proteases and the application of these tools with new experimental methods to improve our understanding of proteases in human health and disease. Such protease- directed chemical tools drive biological insights, and this is best evidenced by the seminal studies on serine hydrolases by Balls and Jansen with diisopropylfluorophosphate (1952), as well as the use of tosyl phenylalanyl chloromethyl ketone by Shaw and Schoellmann that established the reactive residues in chymotrypsin (1963). Surprisingly, the types of chemistry used, and the proteases targeted have been fairly limited in scope in the majority of protease-directed molecules available. For example, almost all small-molecule and peptide-based probes target the non-prime-side region of the protease active site and the peptide probes typically consist of natural amino acids. These constraints result in promiscuous molecules that prohibit their use in cells where one needs to confidently assign function to individual proteases. Genetic approaches (e.g. CRISPR/Cas9) provide exquisite control of proteins within cells; however, these methods cannot distinguish enzymatic contributions from the potential roles involving protein:protein interactions. Thus, there is an urgent need for selective small molecules and peptides that spatially and temporally detect and regulate individual proteases within cells. We have made significant advances in the chemistry used for protease-directed molecules, as well as in the types of proteases studied. Our innovative use of unnatural amino acids and unique warheads resulted in the first peptides with selectivity to individual human caspases. We have also made non-hydrolyzable peptides that extend into the prime-side of the active site and have developed assays to discover procaspase activators and inhibitors. We anticipate designing specific molecules for four human caspases within the next five years to address major outstanding questions, including what are the cellular substrates for specific caspases and what are the differences in substrates during apoptosis and T cell activation. We have expanded our probe and small molecule designs to interrogate proteases in pathogenic and commensal bacteria in combination with structural and biochemical methods. We plan to establish bacterial lipoprotein signal peptidases as targets for novel antibiotics and identify how protease inhibition affects expression of surface-bound bacterial proteins. Proteases secreted by gut commensal bacteria directly influence host homeostasis and represent an entire new set of enzymes to be characterized. Our goal is to establish commonalities and differences among highly conserved bacterial proteases and develop chemical probes to establish if aberrant proteolytic activity is a driver of disease; for example, our studies show dramatic elevated bacterial protease levels in the gut during colitis. Our results and methods will enable an unprecedented evaluation of individual proteases during cellular events at resolutions not previously possible and can be extrapolated to include other proteases and biological processes.